| Cepko/Tabin Lab ,Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/V2.html
Transient transfection protocol for the ecotropic/amphotropic producer line from Gary Nolan including titering and concentration of viral stocks.
Solutions
2X HBS8.0 g NaCl
6.5 g HEPES (Sigma H-7006)
10 ml Na2HPO4 (5.25 g sodium phosphate dibasic in 500 ml Q)
2M CaCl
2
29.4 g CaCl
up to 100 ml with Q
2 (Mallinckrodt #4160)filter sterilize and store in aliquotes at -20°
50 mM Chloroquine
2.57 g chloroquine (Sigma #C6628)
up to 100 ml with Q
filter sterilize and store in aliquotes at -20°
1000X Polybrene
filter sterilize and store in aliquotes at -20°
1000X Diptheria Toxin5 mg diptheria toxin (Sigma)
up to 5 ml with 1X PBS
filter sterilize and store in aliquotes at -20°
1000X Hygromycin
filter sterilize and store in aliquotes at -20°
10X PBS
2HPO4
`2.4 g KH
2PO4
up to 1 liter with Q, pH to 7.4 and autoclave
20% Paraformaldehyde/4% Paraformaldehyde-PBS200 g paraformaldehyde
1 ml 10N NaOH
up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°mix 100 ml 20% paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q
filter, and store at 4° for up to 2 weeks
0.5% Gluteraldehyde-PBS
(Nolan says to use 0.05% gluteraldehyde to fix 3T3 for titering)
use immediately
AP Detection Buffer
(for every 7.5 ml add 25 microliters NBT and 19 microliters BCIP)
2
up to 1 liter with QBCIP
0.5 g BCIP (p-toluine salt Sigma #B-8503)
up to 10 ml with DMF
store at -20° in light proof glass container
NBT0.75 g NBT (Sigma #N-6639)
up to 10 ml with 70% DMF
store at -20° in light proof glass container
X-Gal Stain Solution14.7 g Potassium Ferrocyanide 35 mM (Sigma #P-9387)
11.5 g Potassium Ferricyanide 35 mM (Sigma #P-8131)
0.41 g (2 mM) MgCl
2
0.02% NP40 (diluted from a 10% stock)
0.01% Deoxycholic Acid Sodium Salt (Sigma #D-6750)
50 ml 10X PBS
up to 500 ml with Q
store at 4°C in a light proof glass bottle
100X X-Gal
store at -20° in a light proof glass bottle
Procedure
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